Kin28, the TFIIH-Associated Carboxy-Terminal Domain Kinase, Facilitates the Recruitment of mRNA Processing Machinery to RNA Polymerase II

Abstract

The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important. We sought to identify the CTD kinase responsible for capping enzyme targeting. The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase–CTD fusion protein such that capping enzyme can bind in vitro. However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, whilesrb10 or ctk1 deletions do not. Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo. Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable. Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription.

ACKNOWLEDGMENTS

We thank Jeff Corden, Rick Young, Mark Solomon, Fred Winston, David Pellman, David Morgan, and Fred Cross for the gifts of plasmids and yeast strains. We are particularly grateful to Rick Young, Christoph Hengartner, and Sang Seok Koh for Srb10-Srb11 and Kin28-Ccl1 protein preparations. We also thank Toshimitsu Takagi and Yasutaka Takase for the generation of Cet1 antibody, Ben Blancowe for the B3 antibody, and Patrick O'Connor for Pta1 antibody.

This work was supported by grants from NIH to S.B., C.L.M., and A.G. S.B. gratefully acknowledges support from the Pew Scholars Program and an American Cancer Society Junior Faculty Research Award.